Enhanced “Off–On” Electrochemiluminescent Biosensor Based on a Multivalent Aptamer-Induced Spatial Confinement Strategy for Ultrasensitive Detection of Membrane Protein (PTK-7)

"Off-on" electrochemiluminescence (ECL) techniques have garnered considerable interest in the biosensing field owing to its high sensitivity, low background signal, high signal-to-noise ratio, and avoidance of false-positive signals. However, a significant hurdle that prevents its further application is the lack of nontoxic, label-free, and easily synthesized ECL luminophores. In addition, achieving high quenching efficiency on these luminophores still requires strategy renewal. In this study, we propose novel Tb-DNA nanoparticles (NPs) as an emerging luminophore with low environmental risk and easy synthesis, which were synthesized in a facile coordination-driven approach. Taking advantage of the sequence programmability of these luminophores conferred by DNA molecules, we further construct an ultralow background "off-on" ECL platform by anchoring a multivalent aptamer on these nanoparticles, which facilitates close proximity with electron acceptors (e.g., dopamine (DA) oxidized by K₂S₂O₈, DA_Ox) and enables efficient electron transfer between the excited state of Tb-DNA NPs and DA_Ox. Using the membrane protein protein tyrosine kinase-7 (PTK-7) as a sensing target, our "off-on" ECL strategy of spatially confined electron acceptors by multivalent aptamer-anchored Tb-DNA NPs displayed good detection performance, exhibiting a detection range of 90 to 10⁶ cells/mL and a detection limit as low as a single-digit number of cells. This work not only puts forward novel Tb-DNA-based ECL luminophores but also proposes a multivalent aptamer-recognized acceptor strategy with high sensitivity, low background noise, and high specificity, which might hold great potential for ultrasensitive membrane protein detection.